The introduction of immunoassays in the 1960s and 1970s greatly increased the number of analytes amenable to precise and accurate measurement. Radio immunoassays (RIAs) and immunoradiometric (IRMA) assays utilize radioisotopic labeling of either an antibody or a competing antigen to measure an analyte. Detection systems based on enzymes or fluorescent labels were then developed as an alternative to isotopic detection systems. D. L. Bates, Trends in Biotechnology, 5(7), 204 (1987), describes one such method based upon enzyme amplification. In this method a secondary enzyme system is coupled to a primary enzyme label, for example, the primary enzyme can be linked catalytically to an additional system such as a substrate cycle or an enzyme cascade. Enzyme amplification results from the coupling of catalytic processes, either by direct modification or by interaction with the product of the controlling enzyme.
U.S. Pat. No. 4,668,621 describes utilization of an enzyme-linked coagulation assay (ELCA) in an amplified immunoassay using a clotting cascade to enhance sensitivity. The process involves clot formation due to thrombin activated fibrin formation from soluble fibrinogen and labeled solubilized fibrinogen. Amplification of the amount of reportable ligand attached to solid phase is obtained only by combining use of clotting factor conjugates with subsequent coagulation cascade reactions.
Substrate/cofactor cycling is another variation of enzyme-mediated amplification, and is based on the cycling of a cofactor or substrate which is generated by a primary enzyme label. The product of the primary enzyme is a catalytic activator of an amplifier cycle which responds in proportion to the concentration of substrate and hence the concentration of the enzyme label. An example of this type of substrate cycling system is described in U.S. Pat. No. 4,745,054.
Vary et al., Clin Chem., 32, 1696 (1986) describes an enzyme amplification method suited to nucleic acid detection. This method is a strand displacement assay which uses the unique ability of a polynucleotide to act as a substrate label which can be released by a phosphorylase.
Bobrow et al., J. of Immunol. Methods, 125, 279 (1989) discloses a method to improve detection or quantitation of an analyte by catalyzed reporter deposition. Amplification of the detector signal is achieved by activating a conjugate consisting of a detectably labeled substrate specific for the enzyme system, wherein said conjugate then reacts with the analyte-dependent enzyme activation system to form an activated conjugate which deposits wherever receptor for the conjugate is immobilized.
Nucleotide hybridization assays have been developed as a means for detection of specific nucleic acid sequences. U.S. Pat. No. 4,882,269 discloses an amplified nucleic acid hybridization assay in which a target nucleic acid is contacted with a complementary primary probe having a polymeric tail. A plurality of second signal-generating probes capable of binding to the polymeric tail are added to achieve amplified detection of the target nucleic acid. Variations of this methodology are disclosed in PCT Application WO 89/03891 and European Patent Application 204510, which describe hybridization assays in which amplifier or multimer oligonucleotides are hybridized to a single-stranded nucleic acid unit which has been bound to the targeted nucleic acid segment. Signal amplification is accomplished by hybridizing signal-emitting nucleic acid bases to these amplifier and multimer strands. In all of these disclosures amplification is achieved by mechanisms which immobilize additional sites for attachment of signal-emitting probes.
In contrast, the present invention utilizes a fundamentally different concept in achieving signal amplification. In response to analyte, a target nucleic acid sequence is immobilized and replicated using nucleic acid replication techniques. Signal enhancement is achieved by generating and detecting replicates of the target sequence.
U.S. Pat. No. 4,994,368 discloses a nucleic acid hybridization assay which accomplishes detection of polynucleotide analytes by producing replicated copies of a primary polynucleotide sequence. The target sequence of interest is first restricted to provide a free 3' OH end, and then is hybridized to a complementary binding sequence located at the 3' end of two or more template sequences in a single-stranded pattern polynucleotide. Chain extension is performed on the target sequence, and this extension product is then cleaved into fragments which are subsequently hybridized with single-stranded pattern nucleotide. The polymerization, cleavage, rehybridization, polymerization cycle is repeated until a detectable number of copies have been obtained. In a similar vein, PCT application WO 90/0345 describes a nucleic acid detection assay wherein the reporter molecule is an adduct comprising 1) an oligonucleotide probe sequence which is complementary to the targeted site; 2) a primer sequence capable of initiating primer extension; and 3) a sequence segment which is complementary to the primer sequence. As initially added to the test nucleic acid sample, the adduct assumes a hairpin structure which renders the primer inactive. Upon hybridization of the adduct to a target sequence in the sample, however, the adduct becomes activated and its primer sequence becomes available for initiating a primer extension product. The art methods differ from that of Applicants' in that the art uses significantly different and more cumbersome approaches to producing multiple copies of a detectable nucleic acid. Also, these methods are limited to the detection of nucleotide sequences, while Applicants' method is applicable to a wide range of analytes.
The use of RNA as a reporter for immunolocical assays has been described in the literature. WO 87/06270 teaches the use of an RNA capable of being autocatalytically replicated by an RNA-dependent RNA polymerase as a reporter for assaying biopolymers by immunoassay or by nucleic acid probe hybridization.
Similarly WO 91/17442 describes various protein/nucleic acid hybrid probes which can be used to amplify the detectable signal in immunoassays. Signal is amplified by a method comprising first immobilizing an antigenic analyte on a solid substrate, binding to the analyte a protein/nucleic acid hybrid probe comprising a double-stranded RNA T7 polymerase promoter operably connected to either a single-stranded or double-stranded nucleic acid template, removing any unbound probe, transcribing multiple copies of RNA oligomers and detecting and quantifying the transcripts. Template replication is on the order of 10.sup.1 to 10.sup.4 copies per template.
The above methods are useful for enhancing the level of detection of analytes by immunoassay, however, both methods suffer from significant restrictions. For example, both methods rely on the use of RNA-dependent polymerases for nucleic acid replication which gives inherently less amplification than other nucleic acid amplification methods such as Polymerase Chain Reaction (PCR) or Ligase Chain Reaction (LCR), and does not result in a molecularly-defined product. It is well known in the art that PCR, for example, will give amplification on the order of 10.sup.6 to 10.sup.14 copies per target of discreet length. Furthermore, the above methods are not easily adapted to the detection of more than one analyte in a sample.
Sano et al. Science, 258, 120, (1992) describes an antigen detection system, termed Immuno-PCR, in which a specific DNA molecule is used as a reporter. A streptavidin-protein A chimera was used to attach a biotinylated DNA to antigen-monoclonal antibody complex that had been immobilized on microtiter plate wells. A segment of the complexed DNA was amplified by Polymerase Chain Reaction (PCR) and the PCR products were analyzed by gel electrophoresis. This method is limited by the need for multiple reagent additions and extensive washing requirements.